1. Field of the Invention
The present invention relates to a sensor for measuring pyrophosphate in a sample solution conveniently with high sensitivity, and a SNP typing sensor using the same.
2. Background of the Related Art
Pyrophosphate has been known to be prominently involved in intracellular enzymatic reactions. For example, in the process of protein synthesis, pyrophosphate is produced in a reaction to form aminoacyl tRNA from an amino acid via aminoacyl adenylate. In addition, for example, in the process of starch synthesis found in plants and the like, pyrophosphate is produced when ADP-glucose is produced by a reaction of glucose-1-phosphate and ATP. In addition thereto, pyrophosphate has been known to be involved in a variety of enzymatic reactions. Therefore, techniques for quantitatively detecting pyrophosphate are important in analyses of cellular states, the aforementioned enzymatic reactions and the like.
Known conventional methods for measuring pyrophosphate include a chemical method of Grindley et al., (see, Nonpatent Document 1: G. B. Grindley and C. A. Nichel, Anal. Biochem., Vol. 33. p 114 (1970)). However, this method is not preferred since concentrated sulfuric acid is used.
In Patent Document 1 (Japanese Patent Provisional Publication No. S61-12300), three kinds of methods for measuring pyrophosphate are disclosed in which an enzyme is utilized without using a chemical such as concentrated sulfuric acid. Those methods are explained in the following.
In the first method, pyruvate orthophosphate dikinase is allowed to act on pyrophosphate in the presence of phosphoenol pyruvate and adenosine monophosphate. Since pyruvic acid is produced by this reaction, the amount of pyrophosphate can be determined by measuring the amount of pyruvic acid. As the method for measuring the amount of pyruvic acid, two kinds of methods were proposed. One is a method in which when a catalytic action of lactate dehydrogenase is utilized to reduce pyruvic acid with NADH, decrease of NADH is colorimetrically determined. Another is a method in which pyruvate oxidase is allowed to act on the produced pyruvic acid, and generated hydrogen peroxide is lead to a dye to execute colorimetric determination.
In the second method, glycerol-3-phosphate cytidyl transferase is allowed to act on pyrophosphate in the presence of cytidine diphosphate glycerol. Glycerol triphosphate is produced by this reaction. Therefore, by measuring the amount of produced glycerol triphosphate, the amount of pyrophosphate can be determined. As the method for measuring the amount of glycerol triphosphate, two kinds of methods were proposed. One is a method in which when a catalytic action of glycerol-3-phosphate dehydrogenase is utilized to oxidize glycerol triphosphate with NAD(P)+, increase of NAD(P)H is colorimetrically determined. Another is a method in which glycerol-3-phosphate oxidase is allowed to act on the produced glycerol triphosphate, and generated hydrogen peroxide is lead to a dye to execute colorimetric determination of the same.
In the third method, ribitol-5-phosphate cytidyl transferase is allowed to act on pyrophosphate in the presence of cytidine diphosphate ribitol. Since this reaction produces D-ribitol-5-phosphate, measurement of the production amount enables the amount of pyrophosphate to be measured. As the method for measuring D-ribitol-5-phosphate, a method in which ribitol-5-phosphate dehydrogenase is allowed to act in the presence of NAD+ (or NADP+), and increase of NADH (or NADPH) is colorimetrically determined was proposed.
In addition, Patent Document 2 (Japanese Patent Provisional Publication No. 2002-369698) discloses a method in which: pyrophosphate is hydrolyzed into phosphoric acid by pyrophosphatase; thereafter the phosphoric acid is allowed to react with inosine or xanthosine by purine nucleoside phosphorylase; the generated hypoxanthine is oxidized into xanthine by xanthine oxidase, followed by additional oxidization to produce uric acid; and coloring is permitted by a coloring agent using peroxidase for the hydrogen peroxide generated in the oxidation process by xanthine oxidase.
However, since the absorbance or coloring of the sample is measured in these methods for measuring pyrophosphate, use of a comparatively large-scale apparatus including an optical system is required.
Meanwhile, an extension reaction of a nucleic acid is also one of important biological reactions in which pyrophosphate is involved.
In recent years, techniques in connection with genetic information have been extensively developed. In medical field, analyses of a disease-related gene have enabled therapy of the disease at a molecular level. Also, gene diagnoses have enabled tailor-made medicine for every individual patient. In the field of pharmaceutical, genetic information is used to specify protein molecules such as antibodies and hormones, which are utilized as a chemical. Also in the field of agriculture and foods, products have been produced utilizing a lot of genetic information.
Among such genetic information, gene polymorphism is particularly important. As our facial appearances and body figures vary from one another, genetic information of every person also varies in significant parts. Among the differences of the gene information, those exhibiting alteration of the base sequence at a frequency of no less than 1% of the population are referred to as gene polymorphism. Such gene polymorphism has been suggested to be involved in not only facial configuration of each of the individuals, but also causes of a variety of genetic diseases, as well as constitution, pharmaceutical preparation responsiveness, side effects by pharmaceutical preparations, and the like. Currently, relationships between the gene polymorphisms and diseases and the like have been investigated rapidly.
Among these gene polymorphisms, SNP (Single Nucleotide Polymorphism) has particularly attracted attention in recent years. The SNP refers to gene polymorphism including only one different base in the base sequence of genetic information. SNP has been reported to be present within human genomic DNA as many as two to three million, and expected for utilization in clinical field since it can be readily utilized as a marker of gene polymorphism. At present, for the techniques relating to SNP, in addition to researches of identification of the position of a SNP in a genome, and to relationship between SNP and a disease, developments of SNP typing techniques for discriminating the base at a SNP site have been executed.
There are a variety of types of techniques for SNP typing such as those in which hybridization is utilized, those in which a restriction enzyme is utilized, those in which an enzyme such as ligase is utilized, and the like. Among those techniques, the most convenient technique utilizes a primer extension reaction. In this technique, the SNP typing is carried out by determining the occurrence of a primer extension reaction.
For detection in a SNP typing technique in which a primer extension reaction is used, a method including detecting an actual DNA amplification product using a fluorescent dye and a method including using an immobilization probe, as well as a method including detecting pyrophosphate that is a byproduct of nucleic acid synthesis by DNA polymerase have been also proposed. In such methods, pyrophosphate produced along with progress of the primer extension reaction is converted into ATP for detecting the difference of progress of the extension reaction, and thereafter the amount of pyrophosphate is measured utilizing a luciferase reaction (see, Nonpatent Document 2: J. Immunological Method, 156, 55-60, 1992).
Furthermore, Patent Document 3 (Pamphlet of International Patent Publication No. 03/078655) discloses a method in which a target nucleic acid in a sample solution is determined with high sensitivity by measuring pyrophosphate, and a method for conveniently typing a SNP. In Patent Document 3, there is disclosed a method for typing of a SNP sequence of a DNA including: subjecting the sample to a reaction system that includes DNA polymerase, deoxynucleotides, and a DNA probe having a SNP site and having a sequence complementary to a SNP sequence of a DNA; extending the DNA probe by a PCR (Polymerase Chain Reaction) reaction; converting pyrophosphatase produced with the extension of the DNA probe into an inorganic phosphoric acid by pyrophosphatase; and finally measuring the value of an electric current with an electrode using a measurement system that includes glyceraldehyde-3-phosphate, oxidized nicotineamide adenine dinucleotide, glyceraldehyde-3-phosphate dehydrogenase, diaphorase and potassium ferricyanide as an electronic mediator. According to this method, it is mentioned that the SNP sequence can be discriminated within 100 sec after adding the sample including pyrophosphate into the measurement system.
Since the measurement of pyrophosphate and the SNP typing are enabled by electrochemically determining the redox reaction of the electronic mediator in this method, it is disclosed as a method that is highly sensitive and convenient, without need of an optical system.
In such measurement of pyrophosphate and SNP typing, down sizing of the sensor chip is enabled by carrying necessary reagents in a dried state on the sensor substrate, and further, the measurement by a very simple operation can be performed.
Patent Document 4 (Japanese Patent Provisional Publication No. Hei 7-83872) discloses that when a buffer component and an enzyme are concomitantly carried on a biosensor of the reagent-carrying type in a dried state, deterioration of the enzyme activity can be prevented by separately arranging each of them.